Chromatograpny, as it is generally used, is a technique used for the separation of various components of a "sample" mixture. In a liquid chromatography system, a sample followed by an elution fluid are injected into a separation column. The separation column contains a packing or matrix medium or material, as well known in the art, which interacts with the various components of the sample fluid to be separated. The composition of the separating medium depends on the fluid being directed therethrough so as to produce the desired separation. The separation columns generally known in the art are of a cylindrical construction and the fluid flows axially through a separating medium bed (packing or matrix) retained in the column. The medium bed is retained between supports or frits on either or both ends of the column. As the sample and elution fluids pass through the separating medium bed, the constituents of the sample fluid travel at different rates due to their interaction with the matrix or packing material. As a result, these constituents emerge separated (i.e., have different elution times) in the outlet stream of the column.
These prior known approaches are exemplified by the following U.S. Patent No. 3,230,167 issued Jan. 18, 1966 to M. J. E. Golay; U.S. Pat. No. 3,422,605 issued Jan. 21, 1969, to R. P. Crowley; U.S. Pat. No. 3,453,811 issued July 8, 1969, also to R. P. Crowley; U.S. Pat. No. 3,780,866 issued Dec. 25, 1973 to L. V. Ek et al; U.S. Pat. No. 4,133,562, issued Jan. 9, 1979 to L. H. Andren; U.S. Pat. No. 4,350,595 issued Sept. 21, 1982 to W. Gunkel; U.S. Pat. No. 4,354,932 issued Aug. 19, 1982 to R. J. McNeil; and U.S. Pat. No. 4,496,461 issued Jan. 29, 1985 to G. Leeke et al.
In the case of conventional chromatography, the available matrices or separation material beds for separating substances of large molecular weight are soft and compress easily. Matrix compression in turn causes dramatically reduced flow through the separation column. When chromatographic separation systems are scaled up for commercial purposes, more matrix volume is required and thus larger columns have to be employed. Additionally, the process requires a substantial increase in the fluid flow rate to achieve acceptable production rates. The combination of high flow rates and larger bed height (i.e., hydrostatic head) results in high pressure drops across the matrix that in turn further compress the matrix material, adversely affecting flow through the column. Some prior designs have addressed this problem by incorporating short, wide columns; i.e., columns with large cross-sectional area and reduced height. While this prior design does help reduce pressure drops and improve throughput, the geometry results in large saucer shaped (center dipping) columns when additional scale up is desired. Larger diameter columns have the problems of: (1) inconvenient geometry for fabrication, (2) difficulty in even packing of the column, (3) uneven distribution of the sample over the cross-sectional area, and (4) large dead volume leading to loss in chromatographic resolution. Due to these problems, scale up is often accomplished by using multiple columns in parallel or using larger columns but with smaller diameter-to-height ratios. The first alternative mentioned above can be cumbersome and often results in high costs while the second alternative leads to a recurrence of the problem with compression of the matrix or separator material bed. The process has to be reoptimized since the flow rates have to be altered to reduce pressure drop, leading to considerable expense in terms of time and material.
High performance chromatography, on the other hand, has confronted the problems of low flow rates and poor resolution by the use of small size (range of 3-60 um) column packings made from inert, rigid column supports such as silica, glass, polymers, hydroxylapatite, metals etc. The rigid supports are designed to withstand pressures of the order of several thousand psi.
Traditionally, high performance liquid chromatography (HPLC) has been carried out in a manner very similar to conventional chromatography. The column packings are compressed in a long, narrow bed between two bed supports or frits inside a long, narrow column. The sample is applied to the top of the bed and interacts with the packing material in one of a variety of ways such as ion-exchange, absorption, affinity, adsorption, hydrophilic or hydrophobic interactions and by other mechanisms known to those skilled in the art. The degree ano strength of the interaction varies with the type and nature of the packing material and the components of the sample mixture. Thus, when an "eluant" which also interacts with the sample, is passed through the bed, each of the components of the sample mixture dissociates differently, travels down the column at different rates and are thus differentially eluted. The concentration of each component in the effluent or eluant stream may be determined by measuring the amount of a detectable component or label contained in the sample fluid by methods known in the art, which include but is not limited to absorption or emission spectrometry in the uv, visible or infra red range, measurement of the refractive index, radioactivity, fluorescence tagging and the like.
High performance liquid chromatography has been applied to separating complex mixtures, often with components with very similar physical and chemical properties. This nas generally been possible because of the very small size particles used in HPLC. The small particle size of the separating medium gives rise to a large surface area in a given volume which allows for a high degree of surface interactions. This high degree of surface interactions in turn results in low height equivalent per theoretical plate (HETP) and high efficiency high performance separations.
This form of chromatography has been, as discussed earlier, carried out in long, narrow bore columns. The combination of long bed height, and small particle size with high flow rates gives rise to an enormous back pressure which varies from about 50 psi to 5000 psi. These high back pressures drastically limit the choice of preferred column packings. Furthermore, especially in the separation of larger, labile biomolecules , there is a risk of these sheer sensitive proteins or macromolecules being denatured by the high pressures generated. Finally and most importantly, special packing materials, special pumps, special mixers, special injectors and other associated instrumentation designed to withstand these high pressures have had to be developed and utilized. While HPLC enjoys wide spread use because of its high separation potential, especially of closely related compounds, the need for the development of specialized materials and equipment has driven up the price of the column packings, HPLC components and equipment as well as integrated systems.
Efforts to scale up HPLC from an analytical mode to a preparative level require high flow rates and high capacities and have necessitated increasing the column height. This in turn has resulted in even higher back pressures which have proved to be impractical to handle. Widening the column bore has resulted in other types of problems such as uneven distribution of the sample over the entire cross section of the bed and channelling or tunnelling along the wall of the column. These result in decreased resolution, reduction in column capacity and an over all decrease in operating performance. New sample and eluant distributors to overcome uneven distribution, and radial-pak columns to minimize the channelling effect, have been designed and tested. However, the problems associated with difficult scale-up, high flow rates, resulting high back pressures and exorbitant price tags for preparative HPLC are wide spread and are of enormous concern especially as the emerging biotechnology companies scale up production levels.
The above mentioned problems have led to the search for economical column packings which would lower pressure drops, for example, spherical silica-based supports, monodispersed polymeric beads etc. Some of these efforts, including new and different columns, are exemplified by the following patents.
U.S. Pat. No. 4,496,461 issued Jan. 29, 1985 to Leeke et al., and U.S. Pat. No. 4,512,897 to Crowder et al., disclose a unique column design in which fibrous-like packing material is immobilized on membrane sheets. The membrane sheet is spirally wound around a hollow core and is encased in a cartridge. The cartridge is in turn enclosed in a housing. Sample and eluant are introduced into the housing from outside the cartridge, flow through the spirally wound membrane and are collected and removed from the center core. The complex flow pattern, the swellable nature of the matrix and membrane material and the high dead volume results in considerable band broadening and poor separations.
The above design was then modified to a configuration where multiple doughnut shaped disks of membranes are stacked vertically inside the cartridge. This arrangement allows for fast flow and also limits expansion and contraction of the matrix and membrane materials, but the deal volume in the column remains significant, leading to low resolution in the chromatographic separation. Furthermore, the capacity of the column is limited by the nature, amount and size of the particles that could be effectively and efficiently immobilized on the membrane surface. Additionally, the membrane material normally used with these systems are generally unstable in organic solvents often used with HPLC. Thus, these columns are not suitable for application in HPLC.
The copending patent application referenced hereinabove, describes and claims a unique column using horizontal flow chromatography. This column which is described in the referenced patent application employs a design wherein sample fluid is introduced via a distributor to the outer circumferential wall of the bed, travels radially (or horizontally) in through the bed, which consists of packing material, where the components get separated and exit via the collection port in the center of the column. This horizontal flow column has a high cross sectional area and very low effective bed height. It thus offers the ability to handle very hign flow rates at low operative pressures. Since the bed height remains constant even with scale up, the design offers the linear scale-up feature described in the above referenced application. However, the column described in the prior application, specifically the cylindrical embodiment has several drawbacks: (i) too high a dead volume for HPLC applications; (ii) no easy way to pack the column; (iii) no easy way to remove air once it is trapped in the system; (iv) materials of construction not suitable for HPLC applications; (v) too many turns in the distributor channels which would easily be plugged up with debris and would be difficult to clean; and (vi) cumbersome fabrication.
Therefore, it is an object of the present invention to provide a horizontal flow high performance chromatography column.
A further object of the invention is to provide an apparatus for high performance liquid chromatography (HPLC) which overcomes or reduces the above referenced drawbacks of the prior known horizontal flow chromatographic columns.
Another object of the invention is to provide an improved horizontal flow HPLC wherein the dead volume is reduced and the sample flow channels are simplified thereby eliminating or reducing the possibility of plugging.
Another object of the invention is to provide an improved horizontal flow chromatography column which is simplified in construction providing for easy packing of the column and removal of trapped air therefrom.
Additional objects, advantages and novel features of the invention will be set forth in part in the description which follows, and in part will become apparent to those skilled in the art upon examination of the following or may be learned by practice of the invention. The objects and advantages of the invention may be realized and attained by means of the instrumentalities and combinations particularly pointed out in the appended claims.